Diagnostic method for diseases by screening for hepcidin in human or animal tissues, blood or body fluids and therapeutic uses therefor

ABSTRACT

The present invention concerns methods and kits for diagnosing a disease condition characterized by non-physiological levels of hepcidin protein, including prohepcidin and fragments thereof, comprising obtaining a tissue or fluid sample from a subject; contacting the sample with an antibody or fragment thereof that specifically binds to a polypeptide corresponding to the amino acid sequence between and including amino acids 25 and 49 of a hepcidin precursor protein, and quantifying the hepcidin precursor level using an assay based on binding of the antibody and the polypeptide; wherein the non-physiological level of prohepcidin is indicative of the disease condition. The present invention also concerns diagnostic methods and kits for applications in genetic technological approaches, such as for overexpressing or downregulating hepcidin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 10/299,486filed Nov. 19, 2002, now U.S. Pat. No. 7,411,048.

BACKGROUND ART

Iron is an essential trace element that is required indispensable forDNA synthesis and a broad range of metabolic processes. However,disturbances of iron metabolism have been implicated in a number ofsignificant mammalian diseases, including, but not limited to irondeficiency anemia, hemosiderosis or the iron overload diseasehemochromatosis (Andrews, N. C. (2000) Annu. Rev. Genomics Hum. Genet.1,75-98; Philpott, C. C. (2002) Hepatology 35,993-1001; Beutler et al.,(2001) Drug-Metab. Dispos. 29, 495-499). Excess iron accumulation has anadverse effect, as exemplified by patients with hereditaryhemochromatosis, some of whom die at an early age from cirrhosis of theliver, diabetes, and cardiac failure. Beutler et al., (2001) Drug-Metab.Dispos. 29, 495-499. Iron content in mammals is regulated by controllingabsorption predominantly in the duodenum and upper jejunum, and is theonly mechanism by which iron stores are physiologically controlled(Philpott, C. C. (2002) Hepatology 35, 993-1001). Following absorption,iron is bound to circulating transferrin and delivered to tissuesthroughout the body. The liver is the major site of iron storage. There,transferrin-bound iron is taken into the hepatocytes byreceptor-mediated endocytosis via the classical transferring receptor(TfR1) (Collawn et al., (1990) Cell 63, 1061-1072) and presumably ingreater amounts via the recently identified homologous transferrinreceptor 2 (TfR2) (Kawabata et al., (1999) J. Biol. Chem. 274,20826-20832). The extracellular domain of this protein is 45% identicalto the corresponding portion of TfR1 (Id.). TfR2 can also bind diferrictransferrin and facilitate the uptake of iron. Mutations in TfR2 havebeen associated with certain forms of hemochromatosis demonstrating theimportant role for TfR2 in iron homeostasis (Philpott, C. C. (2002)Hepatology 35, 993-1001; Camasehella et al., (2000) Nat. Genet. 25,14-15; Fleming et al., (2002) Proc. Natl. Acad. Sci. USA 99,10653-10658). TfR2 is predominantly expressed in the liver (Fleming etal., (2000) Proc. Natl. Acadi. Sci. USA 97, 2214-2219; Subramaniam etal., (2002) Cell Biochem. Biophys. 36, 235-239), however, the exactcellular localization is still unknown.

A feedback mechanism exists that enhances iron absorption in individualswho are iron deficient, and reduces iron absorption in subjects withiron overload (Andrews, N. C. (2000) Annu. Rev. Genomics Hum. Genet. 1,75-98; Philpott, C. C. (2002) Hepatology 35, 993-1001; Beutler et al.,(2001) Drug-Metab. Dispos. 29, 495-499). Nonetheless, the molecularmechanism by which the intestine responds to alterations in body ironrequirements remains poorly understood. In this context, hepcidin, arecently identified mammalian polypeptide (Krause et al., (2000) FEBSLett. 480, 147-150; Park et al., (2001) J. Biol. Chem. 276, 7806-7810),is predicted as a key signaling component regulating iron homeostasis(Philpott, C. C. (2002) Hepatology 35, 993-1001; Nicolas et al., (2002)Proc. Natl. Acad. Sci. USA 99, 4396-4601). Hepcidin was initiallyisolated as a 25 amino acid (aa) polypeptide in human plasma and urineexhibiting antimicrobial activity (Krause et al., (2000) FEBS Lett. 480,147-150; Park et al., (2001) J. Biol. Chem. 276, 7806-7810). A hepcidincDNA encoding an 83 aa precursor in mice and an 84 aa precursor in ratand man, including a putative 24 aa signal peptide, were subsequentlyidentified searching for liver specific genes that were regulated byiron (Pigeon et al., (2001) J. Biol. Chem. 276, 7811-7819).

Since the discovery that hepcidin expression is abolished in miceexhibiting iron-overload due to the targeted disruption of upstreamstimulatory factor 2 (Usf2) gene resembling the same phenotype as foundin Nicolas, O., Bennoun, M., Devaux, I., Beaumont, C., Grandchamp, B.,Kahn, A. & Vaulont, S. (2001) Proc. Natl. Acad. Sci. USA 98, 8780-8785,it has become evident that this peptide plays a pivotal role in ironmetabolism. In contrast, overexpression of hepcidin was shown to resultin severe iron deficiency anemia in transgenic mice (Nicolas et al.,(2002) Proc. Natl. Acad. Sci. USA 99, 4396-4601), indicating thathepcidin is a central regulator of iron homeostasis. However, themechanism by which hepcidin balances the body iron stores or adjusts thedietary iron absorption still remains to be identified. In this respect,the cellular and subcellular localization of this peptide is of decisiveimportance in the search for the signaling route. Although Northern blotanalysis of human and mouse hepcidin mRNA levels in various organsrevealed that hepcidin is predominantly expressed in liver, no dataexist on the cellular source of this polypeptide (Krause et al., (2000)FEBS Lett. 480, 147-150; Park et al., (2001) J. Biol. Chem. 276,7806-7810; Nicolas et al., (2002) Proc. Natl. Acad. Sci. USA 99,4396-4601).

SUMMARY OF THE INVENTION

The present invention concerns hepcidin regulation of iron uptake bymammalian cells and the use of hepcidin and/or hepcidin specificantibodies in the diagnosis of diseases involving disturbances of ironmetabolism. The diagnostic detection kits of the present invention canbe particularly useful in screening the overall population of eitherhumans or animals and identifying those subjects who have thesediseases.

One aspect of the invention is a method for diagnosing a diseasecondition characterized by non-physiological levels of hepcidin,comprising obtaining a tissue or fluid sample from a subject; contactingthe sample with an antibody or fragment thereof that specifically bindsto a polypeptide from the mid-portion (amino acids 20 to 50 of SEQ IDNO: 2) or C-terminus of hepcidin (amino acids 65 to 84 of SEQ ID NO: 2),and quantifying the hepcidin level using an assay based on binding ofthe antibody and the polypeptide; wherein the non-physiological level ofhepcidin is indicative of the disease condition. In one aspect of thepresent invention, sensitive diagnostic methods and kits wereestablished enabling the detection of pro-hepcidin in human plasma. Theinvention opens a broad range of therapeutic perspectives, where ahepcidin antibody and diagnostic methods and kits can be used for thedetermination of hepcidin as a parameter for the progress of thediseases mentioned above during and after therapy.

One embodiment of the invention concerns the generation and purificationof a hepcidin protein and fragments thereof. Another embodiment of theinvention concerns hepcidin specific antibodies, or fragments orvariants thereof that, in turn, can be used in immunoassays to detect ahepcidin protein in suspected humans or animals.

In another aspect of the invention, the hepcidin diagnostic methods andkits can be used in genetic technological approaches, such as foroverexpressing or downregulating hepcidin.

In still another aspect of the invention, hepcidin can be used intherapeutic treatment of the diseases described herein, by treatingsubjects with hepcidin, and agonists or antagonists of hepcidin. Ironuptake in cells could be modulated by varying the concentration ofhepcidin, inhibiting hepcidin binding to iron or to the TfR2 receptor.Accordingly, hepcidin, and agonists or antagonists of hepcidin may beuseful in the treatment of conditions where there is a disturbance iniron metabolism. For example, such substances may be useful in thetreatment of such aforementioned diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A, B) RT-PCR analysis of human liver (lanes 2 and 3) and HepG2cells (lanes 4 and 5) showing gene expression of hepcidin (A) and TfR2(B) with amplification products of correct molecular size. A bp DNAladder is indicated (lanes 1 and 7). Lanes 6 show a negative control.

(C-E) Western blot analyses of hepcidin in extracts of guinea pig(lanes 1) and human liver (lanes 2) as well as in HepG2 cells (lanes 3)and guinea pig skeletal muscle (lanes 4, control) with antibodiesEG(1)-HepN©, EG(2)-HepN (D) and EG(1)-HepC (E). Note the immunoreactivebands at 10 and 20 kDa obtained with all antibodies recognizingdifferent epitopes in a hepcidin precursor. (F) Western blot analysis ofTfR2 in extracts of mouse liver (1), human liver (2), HepG2 cells (3)and mouse heart (4) (control).

FIG. 2. Cellular localization of hepcidin in guinea pig (A-F) and human(G-I) liver. The paraffin sections immunostained with theregion-specific antibodies EG(1)-HepN (A, D, G), EG(2)-HepN (B, E, H)and EG(1)-HepC (C, F, I) show a distinct immunoreactivity at thebasolateral membrane domain of hepatocytes (arrows). (Magnification:A-C, ×180; D-I, ×540).

FIG. 3. Immunohistochemical sections (A, antibody EG(1)-HepN; B,antibody EG(2)-HepN: C, antibody EG(1)-HepC showing the clear zonationof hepcidin within the hepatic lobules with decreasing immunoreactivityfrom periportal zones (stars) towards the central veins (arrowheads).Note that no immunoreactivity is found in hepatocytes around the centralveins. (The arrow in B indicates a portal triad.) (A-C, ×180).

FIG. 4. Immunohistochemical localization of TfR2 in mouse (A-C) andhuman liver (D) using the antibody BT-TFR21-S. Note thatimmununoreactivity is exclusively confined to the basolateral membrane(arrows) of hepatocytes; no immunoreactivity is found around the centralveins (stars in A and C). A slight zonation for TfR2 is seen in A withdecreasing immunoreactivity toward the central vein (A, ×180; B, C,×360; D, ×540).

FIG. 5. Detection of hepcidin (A-C) and TfR2 (D) in HepG2 cells byimmunofluorescence microscopy using the antibodies EG(1)-HepN (A),EG(2)-HepN (B), EG(1)-HepC (C), and BT-TFR21-S (D) (Scale bar 8 mm).

FIG. 6. ELISA for circulating human hepcidin. A representative standardcurve with concentrations of hepcidin-(28-47) (SEQ ID NO: 3) in ng/mland the extinction of the ELISA solution at 450 nm wavelength are shown.Note the high resolving power in the range of 1 to 400 ng/mlhepcidin-(28-47).

FIG. 7 shows the complete nucleotide (SEQ ID NO: 1) and amino acidsequences (SEQ ID NO: 2) of one form of hepcidin reproduced from GenBankdatabase accession nos. NM021175 and AAH20612, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes that hepcidin regulates iron uptake bymammalian cells and nonphysiological express of hepcidin results indisease involved distribution of iron metabolism. The physiologicalconcentration of hepcidin is in the range of 200-260 ng/mL.Nonphysiological concentrations are below or over this range.Nonphysiological amounts of hepcidin protein or fragment thereof areassociated with disturbances of iron metabolism, resulting in irondeficiency or overload, such as iron deficiency anemia; genetic andnongenetic iron overload diseases, such as hemosiderosis andhemochromatosis or secondary hemochromatosis, aceruloplasminemia,hypotransferrinemia, atransferrinemia; iron overload diseases ofundetermined origin, for instance in the case of diseases of the biliarysystem, liver diseases, especially alcoholic liver diseases,nonalcoholic steatohepatitis, and chronic hepatitis B and C infections;diseases of utilization of iron, such as sideroblastic anemia,thalassemia; hematologic diseases, such as leukemia, polyglobulie,macrocytic, microcytic or normocytic anemia, anemia withreticulocytosis, hemolytic anemia; disturbances of thereticuloendothelial system due to infections and diseases; inflammationsand infections, including sepsis; immunologic diseases and tumors, suchas carcinoma, sarcoma, lymphoma, that result in non-physiologic hepcidinconcentrations; neurodegenerative diseases, such as Alzheimer's diseaseand Wilson's disease. The clinical consequences of iron overload includecirrhosis of the liver and hepatocellular cancer, diabetes, heartfailure, arthritis, and hypogonadism. Zhou et al., Proc. Natl. Acad.Sci., 95, 2492-2497 (1998). This discovery has permitted the developmentof assays for a hepcidin protein and fragments thereof and theirsubsequent purification with retention of their native configuration andphysiological activity. The invention is based, in part, on thediscovery that in patients suffering from certain disorders a hepcidinprotein is present in tissue, blood and body fluid of a human or animal.

This invention provides the first demonstration that a hepcidin proteinin subjects of these disorders are present in human or animal tissue,blood and body fluids in concentrations greatly exceeding that found innormal humans or animals that are not subjects of these disorders. Thisis achieved by examining a sample of tissue, blood or body fluid from apatient, and detecting the presence and quantity of hepcidin protein.The detection and quantitative measurement of any hepcidin protein orfragment thereof in tissue, blood or body fluids in accordance with thisinvention is useful in confirming a clinical diagnosis of the diseasesdescribed herein, in affected patients and in following the course ofthe disease. The invention is also useful in monitoring the diseaseduring and subsequent to a period of treatment with agents that arebeing tested for their ability to stabilize, decrease or prevent theoccurrence of such diseases.

For purposes of description only, the invention will be described interms of: (a) generating a hepcidin protein or fragments thereof; (b)generating antibodies that specifically bind a hepcidin protein; (c)diagnostic assays and kits for diagnosing subtyping or monitoring thediseases described herein; (d) methods for over expressing and downregulating hepcidin; and (e) therapeutic treatment of the diseasesdescribed herein.

Production of a Hepcidin Protein

Isolating a Hepcidin Protein from Blood and Body Fluids

For purposes of the present invention the term hepcidin protein isdefined as any mammalian hepcidin polypeptide sharing about 80 percentamino acid sequence identity with the predicted amino acid sequencepublished by Pigeon and co-workers ((2001) J. Biol. Chem. 276,7811-7819). The hepcidin proteins provided herein also include proteinscharacterized by amino acid sequences similar to those of purifiedhepcidin proteins but into which modification are naturally provided ordeliberately engineered. For example, modifications in a hepcidinpeptide or DNA sequences can be made by those skilled in the art usingknown techniques. Modifications of interest in a hepcidin proteinsequences may include the alteration, substitution, replacement,insertion or deletion of a selected amino acid residue in the codingsequence. For example, one or more of the cysteine residues may bedeleted or replaced with another amino acid to alter the conformation ofthe molecule. Techniques for such alteration, substitution, replacement,insertion or deletion are well known to those skilled in the art (see,e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration,substitution, replacement, insertion or deletion retains the desiredactivity of the protein. Regions of a hepcidin protein that areimportant for the protein function can be determined by various methodsknown in the art including the alanine-scanning method which involvedsystematic substitution of single or strings of amino acidswith-alanine, followed by testing the resulting alanine-containingvariant for biological activity. This type of analysis determines theimportance of the substituted amino acid(s) in biological activity.

Production of a hepcidin protein may be accomplished by isolating ahepcidin protein from the tissue, blood or body fluids of humans oranimals suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other such diseases described herein,using standard techniques known by those of skill in the art. Suchtechniques included in the invention also relate to methods forproducing a hepcidin protein comprising growing a culture of host cellsin a suitable culture medium, and purifying a hepcidin protein from thecells or the culture in which the cells are grown.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated hepcidin proteins of the present invention. Forexample, a hepcidin protein can also be produced by chemical synthesisof the amino acid sequence of a hepcidin protein (Pigeon et al., (2001)J. Biol. Chem. 276, 7811-7819), as predicted from the cloning andsequencing of a cDNA coding for a hepcidin protein. This hepcidinprotein sequence information may be utilized to predict the appropriateamino sequence of a fragment of a hepcidin protein to be chemicallysynthesized using standard peptide synthesis methods known in the art.These methods include a solid-phase method devised by R. BruceMerrifield, (Erickson and Merrifield, “Solid-Phase Peptide Synthesis”,in The Proteins, Volume 2, H. Neurath & R. Hill (Eds.) Academic Press,Inc., New York pp. 255-257; Merrifield, (1986) “Solid phase synthesis”,Science, 242:341-347). In the solid-phase method, amino acids are addedstepwise to a growing peptide chain that is linked to an insolublematrix, such as polystyrene beads. A major advantage of this method isthat the desired product at each stage is bound to beads that can berapidly filtered and washed and thus the need to purify intermediates isobviated. All of the reactions are carried out in a single vessel, whicheliminates losses due to repeated transfers of products. This solidphase method of chemical peptide synthesis can readily be automatedmaking it feasible to routinely synthesize peptides containing about 50residues in good yield and purity (Stewart and Young, (1984) Solid PhasePeptide Synthesis, 2^(nd) ed., Pierce Chemical Co.; Tam et al., (1983)J. Am. Chem. Soc., 105:6442). For example, a hepcidin protein fragmentcorresponding to amino acid residues 1 to 50, or 34 to 84 as depicted inFIG. 7 could be synthesized. At the simplest level, commerciallyavailable peptide synthesizers are particularly useful in producingsmall peptides and fragments of a hepcidin protein. Fragments areuseful, for example, in generating antibodies against the nativehepcidin protein.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain one of the isolated hepcidinproteins/peptides of the present invention. These include, but are notlimited to, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography, and immuno-affinity chromatography. See,e.g., Scopes, Protein Purification: Principles and Practice,Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: ALaboratory Manual; Ausubel et al., Current Protocols in MolecularBiology.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a hepcidin protein. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinanthepcidin protein. A hepcidin protein thus purified is substantially freeof other mammalian proteins and is defined in accordance with thepresent invention as an isolated protein.

The sequence of a hepcidin protein may be identified using the Edmandegradation method of protein sequencing. This method sequentiallyremoves one amino acid residue at a time from the amino terminal end ofa peptide for subsequent sequence identification by chromatographicprocedures. See for example, the techniques described in Konigsberg andSteinman, (1977) Strategy and Methods of Sequence Analysis, in Neurathand Hill (eds.), The Proteins (3^(rd) ed.) Vol. 3, pp. 1-178, AcademicPress. In addition, sequence analysis of a hepcidin protein may beaccelerated by using an automated liquid phase amino acid sequenatorfollowing described techniques (Hewick et al., (1981) J. Biol. Chem.,256:7990-7997; Stein and Undefriend, (1984) Analy. Chem., 136:7-23),thereby allowing for the analysis of picomolar quantities of a hepcidinprotein.

The purified hepcidin protein can be used in in vitro binding assaysthat are well known in the art to identify molecules that bind to ahepcidin protein. These molecules include but are not limited to, fore.g., small molecules, molecules from combinatorial libraries,antibodies or other proteins. The molecules identified in the bindingassay are then tested for agonist or antagonist activity in in vivotissue culture or animal models that are well known in the art. Inbrief, the molecules are titrated into a plurality of cell cultures oranimals and then tested for either cell/animal death or prolongedsurvival of the animal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to a tumor or other cellby the specificity of the binding molecule for a hepcidin protein.

Cloning and Expression of Recombinant Hepcidin Protein

In other embodiments, production of a hepcidin protein can be achievedby recombinant DNA technology. For example, appropriate hepcidinnucleotide coding sequences may be synthesized, cloned and expressed inappropriate host cells. Since the DNA sequence coding for a hepcidinprotein is known (Pigeon et al., (2001) J. Biol. Chem. 276, 7811-7819),DNA probes may be synthesized by standard methods known in the art toscreen cDNA libraries prepared from liver tissue from human or animalsubjects suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other diseases described herein, forspecific hepcidin protein cDNA's. These DNA probes can further be usedto isolate the entire family of hepcidin protein genes from these cDNAlibraries using methods that are well known to those skilled in the art.See, for example, the techniques described in Maniatis et al., (1982)Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., Chapter 7.

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample that includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. By using stringent hybridizationconditions directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific DNAclone by the hybridization of the target DNA to that single probe in themixture which is its complete complement (Wallace, et al., (1981)Nucleic Acids Research, 9:879).

Alternatively, an expression library can be screened indirectly for ahepcidin protein of the invention having at least one epitope usingantibodies to the protein. Such antibodies can both be polyclonally ormonoclonally derived and used to detect an expression product indicativeof the presence of a hepcidin protein. Generally, a lambda gt11 libraryis constructed and screened immunologically according to the method ofHuynh, et al., (1985) (in DNA Cloning: A Practical Approach, D. M.Glover, ed., 1:49).

The development of specific DNA sequences encoding a hepcidin proteincan also be obtained by: (1) isolation of a double stranded DNA sequencefrom the genomic DNA, and (2) chemical manufacture of a DNA sequence toprovide the necessary codons for the protein of interest.

The polymerase chain reaction (PCR) technique can be utilized to amplifythe individual members of a hepcidin family for subsequent cloning andexpression of hepcidin protein cDNAs (e.g., see U.S. Pat. Nos.4,683,202; 4,683,195; 4,889,818; Gyllensten et al., (1988) Proc. Nat'lAcad. Sci. USA, 85:7652-7656; Ochman et al., (1988) Genetics,120:621-623; Triglia et al., (1988) Nucl. Acids. Res., 16:8156; Frohmanet al., (1988) Proc. Nat'l Acad. Sci. USA, 85:8998-9002; Loh et al.,(1989) Science, 243:217-220).

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing a hepcidin protein or fragmentsthereof coding sequences and appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See, for example, the techniques described in Maniatis etal., 1982, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y., Chapter 12.

A variety of host-expression vector systems may be utilized to express ahepcidin protein or fragment thereof. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a coding sequence for a hepcidin protein or fragmentsthereof; yeast transformed with recombinant yeast expression vectorscontaining a coding sequence for a hepcidin protein or fragment thereof;insect cell systems infected with recombinant virus expression vectors(e.g., baculovirus) containing a coding sequence for a hepcidin proteinor fragment thereof; or animal cell systems infected with recombinantvirus expression vectors (e.g., adenovirus, vaccinia virus) containing acoding sequence for a hepcidin protein or fragment thereof.

The expression elements of these vectors vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in mammalian cell systems, promoters such as theadenovirus late promoter or the vaccinia virus 7.5K promoter may beused. Promoters produced by recombinant DNA or synthetic techniques mayalso be used to provide for transcription of the inserted codingsequence for a hepcidin protein or fragment thereof.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For reviews see, Current Protocols in MolecularBiology, Vol. 2, (1988) Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience Ch. 13; Grant et al., (1987) Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, (1987)Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, (1986) DNA Cloning,Vol. II, IRL Press, Wash., D.C. Ch. 3; and Bitter, (1987) HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology ofthe Yeast Saccharomyces, (1982) Eds. Strathern et al., Cold SpringHarbor Press, Vols. I and II. For complementation assays in yeast, cDNAsfor hepcidin proteins or fragments thereof may be cloned into yeastepisomal plasmids (YEp) that replicate autonomously in yeast due to thepresence of the yeast 2 mu circle. A hepcidin protein or fragmentthereof sequence may be cloned behind either a constitutive yeastpromoter such as ADH or LEU2 or an inducible promoter such as GAL(Cloning in Yeast, Ch. 3, R. Rothstein (1986) In DNA Cloning Vol. 11, APractical Approach, Ed. D M Glover, IRL Press, Wash., D.C.). Constructsmay contain the 5′ and 3′ non-translated regions of a cognate hepcidinprotein mRNA or those corresponding to a yeast gene. YEp plasmidstransform at high efficiency and the plasmids are extremely stable.Alternatively vectors may be used which promote integration of foreignDNA sequences into the yeast chromosome.

A particularly good expression system that could be used to express ahepcidin protein or fragments thereof is an insect system. In one suchsystem, Autographa californica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. A hepcidin protein or fragment thereof coding sequencemay be cloned into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of the polyhedringene results in production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (e.g., see Smith et al.,(1983) J. Biol., 46:586; Smith, U.S. Pat. No. 4,215,051). In addition,materials and methods for baculovirus/insect cell expression systems arecommercially available in kit form from, e.g., Invitrogen, San Diego,Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in theart, as described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987), incorporated herein by reference. Asused herein, an insect cell capable of expressing a hepcidinpolynucleotide of the present invention is transformed.

In cases where an adenovirus is used as an expression vector, a hepcidinprotein or fragment thereof coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vivo or in vitro recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing a hepcidin protein of fragment thereof in infected hosts.(e.g., See Logan & Shenk, (1984) Proc. Natl. Acad. Sci., (USA)81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used.(e.g., see Mackett et al., (1982) Proc. Natl. Acad. Sci., (USA)79:7415-7419; Mackett et al., (1984) J. Virol., 49:857-864; Panicali etal., (1982) Proc. Natl. Acad. Sci., 79: 4927-4931).

Specific initiation signals may also be required for efficienttranslation of the inserted hepcidin protein or fragment thereof codingsequences. These signals include the ATG initiation codon and adjacentsequences. In cases where the entire hepcidin protein genome, includingits own initiation codon and adjacent sequences, are inserted into theappropriate expression vectors, no additional translational controlsignals may be needed. However, in cases where only a portion of ahepcidin protein coding sequence is inserted, exogenous translationalcontrol signals, including the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of a hepcidin protein or fragment thereof coding sequence toensure translation of the entire insert. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bitter et al., (1987) Methods inEnzymol., 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression driven by certainpromoters can be elevated in the presence of certain inducers, (e.g.,zinc and cadmium ions for metallothionein promoters). Therefore,expression of the genetically engineered hepcidin protein or fragmentthereof may be controlled. This is important if the protein product ofthe cloned foreign gene is lethal to host cells. Furthermore,modifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed.

The host cells which contain a hepcidin protein or fragment thereofcoding sequence and which express the biologically active hepcidinprotein or fragment thereof gene product may be identified by at leastfour general approaches: (a) DNA-DNA hybridization; (b) the presence orabsence of “marker” gene functions; (c) assessing the level oftranscription as measured by expression of hepcidin protein mRNAtranscripts in host cells; and (d) detection of hepcidin protein geneproducts as measured by immunoassays or by its biological activity.

In the first approach, the presence of a hepcidin protein or fragmentthereof coding sequence inserted in the expression vector can bedetected by DNA-DNA hybridization using probes comprising nucleotidesequences that are homologous to a hepcidin protein coding sequence orparticular portions thereof substantially as described recently (Pigeonet al., (2001) J. Biol. Chem. 276, 7811-7819).

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if a hepcidin protein or fragment thereof coding sequence is insertedwithin a marker gene sequence of the vector, recombinants containing ahepcidin protein or fragment thereof coding sequence can be identifiedby the absence of the marker gene function. Alternatively, a marker genecan be placed in tandem with a hepcidin protein or fragment thereofcoding sequence under the control of the same or different promoter usedto control the expression of a hepcidin coding sequence. Expression ofthe marker in response to induction or selection indicates expression ofa hepcidin protein coding sequence.

In the third approach, transcriptional activity for a hepcidin proteinor fragment thereof coding region can be assessed by hybridizationassays. For example, RNA can be isolated and analyzed by Northern blotusing a probe homologous to a hepcidin protein or fragment thereofcoding sequence or particular portions thereof substantially asdescribed (Pigeon et al., (2001) J. Biol. Chem. 276, 7811-7819).Alternatively, total nucleic acids of the host cell may be extracted andassayed for hybridization to such probes.

In the fourth approach, the expression of a hepcidin protein or fragmentthereof product can be assessed immunologically, for example by Westernblots, immunoassays such as radioimmunoprecipitation, enzyme-linkedimmunoassays and the like.

Once a recombinant that expresses a hepcidin protein or fragment thereofis identified, the gene product should be analyzed. This can be achievedby assays based on the physical, immunological or functional propertiesof the product. For example, the methods of the invention include aprocess for producing a hepcidin protein in which a host cell containinga suitable expression vector that includes a hepcidin polynucleotide ofthe invention is cultured under conditions that allow expression of theencoded hepcidin protein. A hepcidin protein can be recovered from theculture, conveniently from the culture medium, or from a lysate preparedfrom the host cells and further purified. Preferred embodiments includethose in which the protein produced by such process is a full length ormature form of the protein.

The present invention further provides isolated hepcidin protein encodedby the nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments that differ from anucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical protein sequence. Preferred nucleic acid fragmentsof the present invention are the Orbs that encode proteins.

A hepcidin protein of the present invention can alternatively bepurified from cells that have been altered to express a hepcidinprotein. As used herein, a cell is altered to express a desiredpolypeptide or protein when the cell, through genetic manipulation, ismade to produce a hepcidin protein which it normally does not produce orwhich the cell normally produces at a lower level. One skilled in theart can readily adapt procedures for introducing and expressing eitherrecombinant or synthetic sequences into eukaryotic or prokaryotic cellsin order to generate a cell which produces a hepcidin protein of thepresent invention.

A hepcidin protein of the invention may also be expressed as a productof transgenic animals, e.g., as a component of the milk of transgeniccows, goats, pigs, or sheep which are characterized by somatic or germcells containing a nucleotide sequence encoding a hepcidin protein.

A hepcidin protein may also be produced by known conventional chemicalsynthesis. Methods for constructing a hepcidin protein of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed hepcidin protein sequences, by virtue ofsharing primary, secondary or tertiary structural and/or conformationalcharacteristics with natural hepcidin protein may possess biologicalproperties in common therewith, including protein activity. Thus, theymay be employed as biologically active or immunological substitutes fora natural, purified hepcidin protein in screening of therapeuticcompounds and in immunological processes for the development ofantibodies.

A hepcidin protein of the invention may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant protein. The resulting expressed hepcidin protein may thenbe purified from such culture (i.e., from culture medium or cellextracts) using known purification processes, such as gel filtration andion exchange chromatography. The purification of a hepcidin protein mayalso include an affinity column containing agents which will bind to theprotein; one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GASepharose™; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

Alternatively, a hepcidin protein of the invention may also be expressedin a form that will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. A hepcidinprotein can also be tagged with an epitope and subsequently purified byusing a specific antibody directed to such epitope. One such epitope(“FLAG®”) is commercially available from Kodak (New Haven, Conn.).

Other fragments and derivatives of the sequences of hepcidinproteins/peptides which would be expected to retain protein activity inwhole or in part (e.g., binding to a TfR2 receptor, binding to ahepcidin specific antibody, etc.) and are useful for screening or otherimmunological methodologies may also be easily made by those skilled inthe art given the disclosures herein. Such modifications are encompassedby the present invention.

A hepcidin protein or fragment thereof should be immunoreactive whetherit results from the expression of the entire gene sequence, a portion ofthe gene sequence or from two or more gene sequences which are ligatedto direct the production of chimeric proteins. This reactivity may bedemonstrated by standard immunological techniques, such asradioimmunoprecipitation, radioimmune competition, or immunoblots.

Generation of Antibodies that Define a Hepcidin Protein

Various procedures known in the art may be used for the production ofantibodies to the mid-portion (amino acids 20 to 50) or C-terminus ofepitopes (amino acids 65 to 84) of a hepcidin protein of SEQ ID NO: 2.The hepcidin specific antibodies bind those epitopes and no other knownsequences. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments and an Fab expressionlibrary. For the production of antibodies, various host animals may beimmunized by injection with a particular hepcidin protein, or asynthetic hepcidin protein, including but not limited to rabbits, mice,rats, etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats. Briefly, hepcidin is utilized toimmunize the animal through intraperitoneal, intramuscular, intraocular,or subcutaneous injections, an adjuvant such as Freund's complete orincomplete adjuvant. Following several booster immunizations, samples ofserum are collected and tested for reactivity to hepcidin. Particularlypreferred polyclonal antisera will give a signal on one of these assaysthat is at least three times greater than background. Once the titer ofthe animal has reached a plateau in terms of its reactivity to hepcidin,larger quantities of antisera may be readily obtained either by weeklybleedings, or by exsanguinating the animal.

Monoclonal antibodies to peptides of hepcidin may be prepared by usingany technique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohler and Milstein,(Nature, (1975) 256:495-497), the more recent human B-cell hybridomatechnique (Kosbor et al., (1983) Immunology Today, 4:72) and theEBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention monoclonal antibodies specific to hepcidinproteins/peptides may be produced in germ-free animals utilizing recenttechnology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote at al., (1983) Proc. Natl. Acad. Sci., 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., (1985)in, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., (1984) Proc. Natl.Acad. Sci., 8 1:6851-6855; Neuberger et al., (1984) Nature, 312:604-608;Takeda et al., (1985) Nature, 314:452-454) by splicing the genes from amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activitycan be used; such antibodies are due to this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce hepcidin protein-specific single chain antibodies.

An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,(1989) Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity to hepcidinproteins/peptides.

Antibody fragments that contain specific binding sites for a hepcidinprotein may be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments.

Diagnostic Assays and Kits

Yet another purpose of the present invention is to provide reagents foruse in diagnostic assays for the detection of a hepcidin protein fromindividuals suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and such other diseases described herein.

In one mode of this embodiment, a hepcidin protein of the presentinvention may be used as an antigen in immunoassays for the detection ofthose individuals suffering from hemochromotosis, iron deficiencyanemia, hemosiderosis, liver cirrhosis and such other diseases describedherein. A hepcidin protein, polypeptide and/or peptide of the presentinvention may be used in any immunoassay system known in the artincluding, but not limited to: radioimmunoassays, enzyme-linkedimmunosorbent assay, “sandwich” assays, precipitin reactions, geldiffusion immunodiffusion assays, agglutination assays, fluorescentimmunoassays, protein A immunoassays and immunoelectrophoresis assays,to name but a few U.S. Pat. No. 4,629,783 and patents cited therein alsodescribe suitable assays.

According to the present invention, monoclonal or polyclonal antibodiesproduced to various forms of a hepcidin protein, can be used in animmunoassay on samples of blood, spinal fluid or other body fluid todiagnose subjects with hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other diseases described herein.

In one embodiment of the invention, a sample of blood is removed fromthe patient by venesection and placed in contact with an anticoagulantsuch as EDTA, mixed, centrifuged at 600 g for 10 min and the plasmaremoved as is common in the art or a sample of spinal fluid is removedfrom the patient by lumbar puncture.

The antibodies described herein may be used as the basic reagents in anumber of different immunoassays to determine the presence of a hepcidinprotein in a sample of tissue, blood or body fluid. Generally speaking,the antibodies can be employed in any type of immunoassay, whetherqualitative or quantitative. This includes both the two-site sandwichassay and the single site immunoassay of the non-competitive type, aswell as in traditional competitive binding assays.

Particularly preferred, for ease of detection, and its quantitativenature, is the sandwich or double antibody assay, of which a number ofvariations exist, all of which are intended to be encompassed by thepresent invention.

For example, in a typical forward sandwich assay, unlabeled antibody isimmobilized on a solid substrate, e.g., microtiter plate wells, and thesample to be tested is brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen binary complex, a secondantibody, labelled with a reporter molecule capable of inducing adetectable signal, is then added and incubation is continued allowingsufficient time for binding with the antigen at a different site and theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, which may be quantitated bycomparison with a control sample containing known amounts of antigen.Variations on the forward sandwich assay include the simultaneous assay,in which both sample and antibody are added simultaneously to the boundantibody, or a reverse sandwich assay in which the labelled antibody andsample to be tested are first combined, incubated and added to theunlabelled surface bound antibody. These techniques are well known tothose skilled in the art, and the possibility of minor variations willbe readily apparent. As used herein, “sandwich assay” is intended toencompass all variations on the basic two-site technique.

For the sandwich assays of the present invention, the only limitingfactor is that both antibodies have different binding specificities fora hepcidin protein. Thus, a number of possible combinations arepossible.

As a more specific example, in a typical forward sandwich assay, aprimary antibody is either covalently or passively bound to a solidsupport. The solid surface is usually glass or a polymer, the mostcommonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinylchloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs or microplates, or any othersurfaces suitable for conducting an immunoassay. The binding processesare well known in the art. Following binding, the solid phase-antibodycomplex is washed in preparation for the test sample. An aliquot of thebody fluid containing a hepcidin protein to be tested is then added tothe solid phase complex and incubated at 25° C. for a period of timesufficient to allow binding of any hepcidin protein present to theantibody specific for hepcidin protein. The second antibody is thenadded to the solid phase complex and incubated at 25° C. for anadditional period of time sufficient to allow the second antibody tobind to the primary antibody-antigen solid phase complex. The secondantibody is linked to a reporter molecule, the visible signal of whichis used to indicate the binding of the second antibody to any antigen inthe sample. By “reporter molecule”, as used in the present specificationis meant a molecule which by its chemical nature, provides ananalytically detectable signal which allows the detection ofantigen-bound antibody. Detection must be at least relativelyquantifiable, to allow determination of the amount of antigen in thesample, this may be calculated in absolute terms, or may be done incomparison with a standard (or series of standards) containing a knownnormal level of antigen.

The most commonly used reporter molecules in this type of assay areeither enzymes or fluorophores. In the case of an enzyme immunoassay anenzyme is conjugated to the second antibody, often by means ofglutaraldehyde or periodate. As will be readily recognized, however, awide variety of different conjugation techniques exist, which are wellknown to the skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase,among others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidineare commonly used. It is also possible to employ fluorogenic substrates,which yield a fluorescent product rather than the chromogenic substratesnoted above. In all cases, the enzyme-labelled antibody is added to thefirst antibody-hepcidin protein complex and allowed to bind to thecomplex, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the tertiarycomplex of antibody-antigen-labeled antibody. The substrate reacts withthe enzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an evaluation of the amount of antigenthat is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein or rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.As in the enzyme immunoassay (EIA), the fluorescent-labelled antibody isallowed to bind to the first antibody-hepcidin protein complex. Afterwashing the unbound reagent, the remaining ternary complex is thenexposed to light of the appropriate wavelength, and the fluorescenceobserved indicates the presence of the antigen. Immunofluorescence andEIA techniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotopes, chemiluminescent or bioluminescentmolecules may also be employed. It will be readily apparent to theskilled artisan how to vary the procedure to suit the required use.

Alternatively, the sample to be tested either human blood or spinalfluid containing a hepcidin protein may be used in a single siteimmunoassay wherein it is adhered to a solid substrate either covalentlyor noncovalently. An unlabeled anti-hepcidin protein antibody is broughtinto contact with the sample bound on the solid substrate. After asuitable period of incubation, for a period of time sufficient to allowformation of an antibody-antigen binary complex a second antibody,labelled with a reporter molecule capable of inducing a detectablesignal, is then added and incubation is continued allowing sufficienttime for the formation of a ternary complex of antigen-antibody-labeledantibody. For the single site immunassay, the second antibody may be ageneral antibody (i.e., zenogeneic antibody to immunoglobulin,particularly anti-(IgM and IgG) linked to a reporter molecule) that iscapable of binding an antibody that is specific for a hepcidin proteinof interest.

A hepcidin gene (mutated or normal) can be utilized in an assay of ironmetabolism. The gene is expressed, with or without any accompanyingmolecules, in cell lines or primary cells derived from human or animalsubjects, healthy subjects, or cells from other organisms (such asrodents, insects, bacteria, amphibians, etc.). Uptake of iron by thesecells is measured, for example through the use of radioactive isotopes.Further, binding of iron to a hepcidin gene product can also bemeasured. Such experiments assist in assessing the role of a hepcidingene and hepcidin gene product in iron uptake, binding, and transport byand in cells.

Therapeutic Treatment

In one aspect of the invention, the hepcidin diagnostic methods and kitscan be used in genetic technological approaches, such as for overexpressing or down regulating hepcidin. In certain therapeuticapplications, it is desirable to down regulate the expression and/orfunction of a hepcidin gene, a mutant hepcidin gene, a hepcidin protein,or a mutant hepcidin protein. For example, down regulation of a normalhepcidin gene or a normal hepcidin protein is desirable in situationswhere iron is under accumulated in the body, for example in certainanemias (i.e., thalassaemias, hemolytic anemias, transfusions). On theother hand, down regulation of a mutant hepcidin gene or a hepcidinprotein is desirable in situations where iron is over accumulated in thebody.

As discussed above antibodies specific to a normal or a mutant hepcidinprotein can be prepared. Such antibodies can be used therapeutically inthe diseases described herein. For example, to block the action of amutant or normal hepcidin gene if the function associated with a mutantprotein is an up regulation of a normal hepcidin protein function andleads to an over accumulation of iron in the body. Similarly, antibodiescan be used therapeutically to block action of a hepcidin protein thatis causing an under accumulation of iron in the body.

In a similar manner, a hepcidin gene, either in a normal or in a mutantform, can be down regulated through the use of antisenseoligonucleotides directed against the gene or its transcripts. A similarstrategy can be utilized as discussed above in connection withantibodies. For a particularly valuable review of the designconsiderations and use of antisense oligonucleotides, see Uhlmann etal., (1990) Chemical Reviews 90:543-584, the disclosure of which ishereby incorporated by reference. The antisense oligonucleotides of thepresent invention may be synthesized by any of the known chemicaloligonucleotide synthesis methods. Such methods are generally described,for example, in Winnacker Chirurg (1992) 63:145. Antisenseoligonucleotides are most advantageously prepared by utilizing any ofthe commercially available, automated nucleic acid synthesizers. Onesuch device, the Applied Biosystems 380B DNA Synthesizer, utilizesbeta-cyanoethyl phosphoramidite chemistry.

Since the complete nucleotide synthesis of DNA complementary to ahepcidin gene is known, the mRNA transcript of the cDNA sequence is alsoknown. As such, antisense oligonucleotides hybridizable with any portionof such transcripts may be prepared by oligonucleotide synthesis methodsknown to those skilled in the art. While any length oligonucleotide maybe utilized in the practice of the invention, sequences shorter than 12bases may be less specific in hybridizing to the target mRNA, may bemore easily destroyed by enzymatic digestion, and may be destabilized byenzymatic digestion. Hence, oligonucleotides having 12 or morenucleotides are preferred. Long sequences, particularly sequences longerthan about 40 nucleotides, may be somewhat less effective in inhibitingtranslation because of decreased uptake by the target cell. Thus,oligomers of 12-40 nucleotides are preferred, more preferably 15-30nucleotides, most preferably 18-26 nucleotides. Sequences of 18-24nucleotides are most particularly preferred.

In still another aspect of the invention, hepcidin can be used in thetherapy of the disorders described herein, by treating subjects withhepcidin, and agonists or antagonists of hepcidin. Iron uptake in cellscan be modulated by varying the concentration of hepcidin, and/orinhibiting hepcidin binding to iron or to the transferrin receptor.Accordingly, hepcidin, and agonists or antagonists of hepcidin may beuseful in the treatment of conditions where there is a disturbance iniron metabolism. For example, such substances may be useful in thetreatment of conditions such as haemochromatosis, neurodegenerativediseases, ischemic tissue damage, including ischemic stroke or trauma,heart disease, and tumors, in particular skin cancer and such otherdiseases described herein. Some have evidence of the end organ injuryseen with prolonged iron overload including liver cirrhosis, arthritis,diabetes, and hypogonadism. Fleming et al., Proc. Natl. Acad. Sci. USA99, 10653-10658 (2002).

The invention also contemplates methods of modulating iron metabolismusing hepcidin. In particular, the present invention relates to a methodfor treating conditions involving disturbances in iron metabolismcomprising administering an iron-modulating amount of hepcidin, or astimulant, agonist or antagonist of hepcidin. Conditions involvingdisturbances in iron metabolism which may be treated using the method ofthe invention include by way of example haemochromatosis,neurodegenerative diseases, ischemic tissue damage, including ischemicstroke or trauma, heart disease, and tumors, in particular skin cancerand such other diseases described herein. A substance which is anagonist or antagonist of hepcidin may be identified by determining theeffect of the substance on the binding activity of hepcidin and iron, orhepcidin and the transferrin receptors TfR1 or TfR2, or the effect ofthe substance on the expression of hepcidin in cells capable ofexpressing hepcidin including cells genetically engineered to expresshepcidin on their surface.

The invention therefore in one aspect relates to a method of identifyingagonists or antagonists of hepcidin comprising reacting a substancesuspected of being an agonist or antagonist of hepcidin with hepcidinand iron under conditions such that hepcidin is capable of binding toiron; measuring the amount of hepcidin bound to iron; and determiningthe effect of the substance by comparing the amount of hepcidin bound toiron with an amount determined for a control. The invention also relatesto a method of identifying agonists or antagonists of hepcidincomprising reacting a substance suspected of being an agonist orantagonist of hepcidin with hepcidin and transferrin receptor underconditions such that hepcidin is capable of binding to the transferrinreceptor; measuring the amount of hepcidin bound to a transferrinreceptor; and determining the effect of the substance by comparing theamount of hepcidin bound to a transferrin receptor with an amountdetermined for a control.

The invention also relates to a method of identifying agonists orantagonists of hepcidin comprising reacting a substance suspected ofbeing an agonist or antagonist of hepcidin with a cell which produceshepcidin, measuring the amount of hepcidin expressed by the cell, anddetermining the effect of the substance by comparing the amount ofexpression of hepcidin with an amount determined for a control. Theinvention further relates to a method for identifying an agonist orantagonist of hepcidin-mediated iron uptake comprising: incubating acell expressing hepcidin on its surface and a substance suspected ofbeing an agonist or antagonist of hepcidin in the presence of iron andin the absence of transferrin, measuring the amount of iron uptake intothe cell, and identifying an agonist or antagonist of hepcidin-mediatediron uptake by comparing the amount of iron uptake in the cell with theamount of iron uptake in a cell from a control incubation in the absenceof the substance.

In some embodiments of the invention, hepcidin peptides are provided fortherapeutic use in subjects having symptoms of a primary iron overloaddisease or syndrome, such as hemochromatosis, or other iron overloadcondition caused by secondary causes, such as repeated transfusions. Ahepcidin peptide can be full-length hepcidin or some fragment ofhepcidin. Preferably, a hepcidin peptide comprises the amino acidresidues 28 to 47 or 70 to 80 of a hepcidin of SEQ ID NO: 2. Thepredicted amino acid sequence and genomic and cDNA sequences of hepcidinwere provided in (Krause et al., (2000) FEBS Lett. 480, 147-150; Pigeonet al., (2001) J. Biol. Chem. 276, 7811-7819), hereby incorporated byreference in their entirety. A hepcidin protein or fragment thereof maybe administered with beta-2-microglobulin, such as in the form of acomplex. In some embodiments, a hepcidin protein greater than about 20amino acids is administered in a complex with beta-2-microglobulin.

In some embodiments of the invention, agonists or antagonists of ahepcidin protein or a transferrin receptor are provided. Agonists of ahepcidin polypeptide, and/or antagonists of a transferrin receptor, areuseful for example, in the treatment of primary or secondary ironoverload diseases or syndromes, while antagonists of a hepcidinpolypeptide, or agonists of the transferrin receptor are useful, forexample, in the treatment of iron deficiency conditions, such asanemias. In other embodiments, mutant hepcidin proteins/peptides areprovided which function as antagonists of the wild-type hepcidinprotein. Antagonists or agonists can also be antibodies, directedagainst a transferrin receptor, or the mid-portion (amino acids 20 to50) or C-terminal region (amino acids 65 to 84) of a hepcidin protein ofSEQ ID NO: 2. In some embodiments of the invention, hepcidinpolypeptides can serve as antagonists of a transferrin receptor. Infurther embodiments of the invention, peptidomimetics can be designedusing techniques well known in the art as antagonists or agonists of ahepcidin protein and/or a transferrin receptor.

Ligands for a transferrin receptor, whether antagonists or agonists, canbe screened using the techniques described herein for the ability tobind to a transferrin receptor. Additionally, competition for hepcidinbinding to a transferrin receptor can be done using techniques wellknown in the art. Ligands, or more generally, binding partners for ahepcidin protein can be screened, for example, for the ability toinhibit the complexing of a hepcidin polypeptide tobeta-2-microglobulin, using techniques described herein.

In some embodiments of the invention, agonists or antagonists oftransferrin are similarly utilized to increase or decrease the amount ofiron transported into a cell, such as into a patient's hepatocytes orlymphocytes. For example, the efficacy of a drug, therapeutic agent,agonist, or antagonist can be identified in a screening program in whichmodulation is monitored in in vitro cell systems. Host cell systems thatexpress various mutant hepcidin proteins/peptides and are suited for useas primary screening systems. Candidate drugs can be evaluated byincubation with these cells and measuring cellular functions dependenton a hepcidin gene or by measuring proper hepcidin protein folding orprocessing. Such assays might also entail measuring receptor-likeactivity, iron transport and metabolism, gene transcription or otherupstream or downstream biological function as dictated by studies ofhepcidin gene function.

Alternatively, cell-free systems can be utilized. Purified hepcidinprotein can be reconstituted into artificial membranes or vesicles anddrugs screened in a cell-free system. Such systems are often moreconvenient and are inherently more amenable to high throughput types ofscreening and automation.

Criteria for the determination of the purity of a hepcidin proteininclude those standard to the field of protein chemistry. These includeN-terminal amino acid determination, one and two-dimensionalpolyacrylamide gel electrophoresis, and silver staining. The purifiedprotein is useful for use in studies related to the determination ofsecondary and tertiary structure, as aid in drug design, and for invitro study of the biological function of the molecule.

In some embodiments of the invention, drugs can be designed to modulatea hepcidin gene and a hepcidin protein activity from knowledge of thestructure and function correlations of a known hepcidin protein. Forthis, rational drug design by use of X-ray crystallography,computer-aided molecular modeling (CAMM), quantitative or qualitativestructure-activity relationship (QSAR), and similar technologies canfurther focus drug discovery efforts. Rational design allows predictionof protein or synthetic structures that can interact with and modify ahepcidin protein activity. Such structures may be synthesized chemicallyor expressed in biological systems. This approach has been reviewed inCapsey et al., Genetically Engineered Human Therapeutic Drugs, StocktonPress, New York (1988). Further, combinatorial libraries can bedesigned, synthesized and used in screening programs.

In order to administer therapeutic agents based on, or derived from, thepresent invention, it will be appreciated that suitable carriers,excipients, and other agents may be incorporated into the formulationsto provide improved transfer, delivery, tolerance, and the like.

A multitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, (15^(th) Edition, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87, by Blaug, Seymour, therein. Theseformulations include for example, powders, pastes, ointments, jelly,waxes, oils, lipids, anhydrous absorption bases, oil-in-water orwater-in-oil emulsions, emulsions carbowax (polyethylene glycols of avariety of molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax.

Any of the foregoing formulations may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive agent in the formulation is not inactivated by the formulationand the formulation is physiologically compatible.

The invention is not limited to the embodiments described herein and maybe modified or varied without departing from the scope of the invention.

EXAMPLES

Tissues and Tissue Preparation

Human liver samples (n=7) used in the present study were obtained afterhemi-hepatectomy in adult subjects with liver metastases. Healthytissues were fixed in 4% paraformaldehyde for immunohistochemistry orimmediately frozen in liquid nitrogen for RT PCR, Western blot andimmunofluorescence analysis.

Guinea pigs (n=7) and mice (n=S) were anesthetized and subsequentlysacrificed by cervical dislocation. Tissue specimens from liver,skeletal muscle and heart were resected and immediately frozen in liquidnitrogen for Western blot analysis or fixed in paraformaldehyde.

Peptide Synthesis, Immunization Procedure, and Antibodies

From the published prohepcidin sequence (Krause et al., (2000) FEBSLett. 480, 147-150; Pigeon et al., (2001) J. Biol. Chem. 276,7811-7819), the peptides hepcidin-(28-47) (SEQ ID NO: 3) andhepcidin-(70-84) (SEQ ID NO: 4) were synthesized as C terminal amidesusing a standard Fmoc protocol (Cetin et al., (1994), Proc. Natl. Acad.Sci. USA 91, 2935-2939) . Peptides were coupled to keyhole limpethemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester, and twoSPF rabbits (Charles River Iffa Credo) were immunized with each peptideconjugate (Eurogentec, Seraing, Belgium). After testing the titer byELISA, three antisera [EG(1)-HepC directed against hepcidin-(70-84) andEG(1)-HepN and EG(2)-HepN, each directed against hepcidin-(28-47) wereused in the present study (hepcidin 28-47: PQQ TGQ LAE LQP QDR AGA RASEQ. (SEQ ID NO: 3), hepcidin 70-84: CGC CHR SKC GMC CKT (SEQ ID NO: 4)). The peptide epitopes used for the generation of the antisera displayedno homology to any hitherto reported protein as confirmed by the BLASTP2 search.

The BT-TFR21 S antibody against mouse TfR2 (BioTrend, Cologne, Germany)was raised against the cytoplasmic N-terminus of mouse TfR2-alpha (TfR2)is alternatively spliced to alpha and beta isoforms, see Fleming et al.,(2000) Proc. Natl. Acad. Sci. USA 97, 2214-2219), showing 68% sequencehomology to the corresponding region of human TfR2-alpha. The antibodywas generated in rabbits and affinity purified.

Expression Analyses in the Human Liver

RNA isolation was performed using Qiagen RNA easy kit including DNAdigestion. Reverse transcription (RT)-PCR analysis was performed asdescribed previously (Kulaksiz et al., (2002) Proc. Natl. Acad. Sci. USA99, 6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664)using the following primers and specifications given in 5-3′orientation: human hepcidin (GenBank database accession no. NM0211175),5′-CTG CAA CCC CAG GAC AGA G-3′ (SEQ ID NO: 5) and 5, GGA ATA AAT AAGGAA GGG AGG GG-3′ (SEQ ID NO: 6), corresponding to nucleotide positions147-165 and 338-316. Human TfR2 (#AF067864), 5′-GAT TCA GGG TCA GGG AGGTG-3′ (SEQ ID NO: 7) and 5′-GAA GGG GOT GTG ATT GAA GG-3′ (SEQ ID NO:8); corresponding to nucleotide positions 2496-2515 and 2694-2675. Afteran initial denaturation of 94° C. for 4 min; reactions were subjected to35 cycles of the following thermal program: 94° C. for 30 s, 60° C. for30 s, and 72° C. for 30 s; this program was followed by a final 5 minelongation step at 72° C. Amplification products were run on an ethidiumbromide-stained 1.8% 89 mM Tris/89 mM boric acid/2 mM EDTA (pH 8.3)agarose gel. The amplification of significant levels of genomic DNA wasexcluded by appropriate controls.

Expression Analyses 1N HepG2 Cells

The human hepatoma HepG2 cells were obtained from the German Collectionof Microorganisms and Cell Culture (Braunschweig, Germany) and grown at37° C. in 5% CO2 in RPMI 1640 media (Gibco, Karlsruhe, Germany)supplemented with 10% (vol/vol) heat-inactivated FBS, penicillin (100units/ml), and streptomycin (100 mg/ml). Cells were analyzed by RT PCRusing the primer specifications mentioned above. For immunofluorescencemicroscopy, HepG2 cells were grown on glass slides fixed for 4 mm inmethanol, and permeabilized with 0.5% Triton X-100 in PBS. Afterincubation with hepcidin (1:2000) and TfR2 antibodies (1:1000) for 60min, followed by incubation with Cy-3-conjugated anti-rabbit antibody(Dianova, Hamburg, Germany), the immunostaining was investigated underan Olympus AX70 microscope using appropriate filters.

Extraction of Hepcidin and TfR2 from Tissues and HepG2 Cells

For hepcidin analysis, frozen tissues and HepG2 cells were mixed in 1 Macetic acid and boiled for 8 mm as described (Cetin et al., (1994),Proc. Natl. Acad. Sci. USA 91, 2935-2939; Cetin et al., (1995) Proc.Natl. Acad. Sci. USA 92, 5925-5929). After homogenization with anUltra-Turrax homogenizer (Janke & Kunkel, Staufen, Germany) the sampleswere centrifuged at 20,000×g for 20 mm at 4° C. and the supernatantswere filtered through a 0.45-mm pore size filter. To enrich proteins,cell and total tissue extracts were applied to an octadecasilyl (C 18)Sep-Pak cartridge (Waters, Mass.). The column was washed with 0.01 M HCland eluted with 30% (vol/vol) 2-propanol/30% (vol/vol) methanol 0.01 MHCl (Cetin et al., (1994), Proc. Natl. Acad. Sci. USA 91, 2935-2939).Protein fractions were lyophilized and stored at −80° C. until use. ForTfR2 analysis, tissues and cells were homogenized in Tris-HCl buffercontaining 100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10% glycerol, 1% TritonX-100, 2 mg/ml leupeptin, 2 mg/ml pepstatin, and 1 mMphenylmethylsulfonyl fluoride, and centrifuged at 100,000 g for 30 mm at4° C.

Immunoblot Analysis

For Western blot analysis, protein extracts were incubated for 7 min at94° C. in sample buffer with 4% (wt/vol) SDS (Merck, Darmstadt,Germany), 50 mM Tris-HCl (pH 8.15), 1 mM EDTA, 3.24 mM dithiothreitol(Roth, Karlsruhe, Germany), 12.5% (wt/vol) glycerol (Merck), and 0.002%bromophenol blue (Merck). To detect hepcidin, a 16.5%tricine-SDS-polyacrylamide gel was used according to the protocolspublished (Cetin et al., (1994), Proc. Natl. Acad. Sci. USA 91,2935-2939; Kulaksiz et al., (2002) Proc. Natl. Acad. Sci. USA 99,6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664; Cetin etal., (1995) Proc. Natl. Acad. Sci. USA 92, 5925-5929). TfR2 immunoblotswere performed using 8% SDS-polyacrylamide gels. Followingelectrophoresis, proteins were transferred onto hydrophobicpolyvinylidene fluoride-based membranes (Pall, Portsmouth, England) bysemidry blotting. The membranes were incubated overnight with hepcidinor TfR2 antibodies at dilutions mentioned above. After washing inTris-buffered saline containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl,and 0.05% Tween 20, the respective immunoreactive proteins werevisualized after incubation with alkaline phosphatase-conjugated goatanti-rabbit antibody (diluted 1:50,000; Sigma) using nirro bluetetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as chromogens(Sigma). The immunoreaction on the Western blot was specifically blockedafter preincubation of the antibodies with the corresponding peptideimmunogens. Crossreactivity with the second goat anti-rabbit antibodywas excluded by appropriate controls (Kulaksiz et al., (2002) Proc.Natl. Acad. Sci. USA 99, 6796-6801; Kulaksiz et al., (2002) Am. J.Pathol. 161, 655-664).

Immunohistochemistry and Immunofluorescence

Tissues were fixed in 4% paraformaldehyde for 18 h at 4° C. Afterdehydration in graded ethanol series, the specimens were embedded inparaffin. Paraffin sections (5 m) were immunostained for hepcidin(antibodies EG(1)-HepN, Kci(2)-HepN, and EG(1)-HepC, each diluted1:2000) or TfR2 (antibody BT-TFR21-S. diluted 1:1000) by theavidin-biotin-peroxidase complex (ABC) technique and incubationsequences as previously described (Kulaksiz et al., (2002) Proc. Natl.Acad. Sci. USA 99, 6796-6801; Kulaksiz et al., (2002) Am. J. Pathol.161, 655-664). The sections were incubated with the respectiveantibodies for 24 h at 4° C., followed by incubation with biotinylatedanti-rabbit IgG (Jackson Immunoresearch, West Grove, Pa., USA) for 30min diluted 1:200. The sections were then incubated for 30 min with apreformed complex of biotin-peroxidase/streptavidin (JacksonImmunoresearch), diluted in PBS (final concentrations:biotin-peroxidase, 0.7 mg/ml; streptavidin, mg/ml). The antigen-antibodybinding sites were visualized by incubation of the sections in 07 mMdiaminobenzidine hydrochloride/0.0020% H₂O₂ in 0.05 M Tris-HCl pH 7.6).

For immunofluorescence microscopy, tissue sections from human liver (2-4mm) were prepared with a cryotome (FrigoCut 2800E; Leica, Nussloch,Germany), air dried for 2 hours, and fixed for 10 min in cold acetone(−20° C.). Double-immunofluorescence labeling was performed as describedpreviously (Rost et al., (1999) Hepatology 29, 814-821) using thespecific hepcidin antibodies (diluted 1:1000) and monoclonal antibodyC219 (id.) raised against canalicular P-glycoproteins (Centocor,Malvern, Pa.) diluted 1:30. After incubation with the respectiveantisera, staining was performed by incubation with Cy2-(1:200) andCy3-(1:600) labeled antibodies against mouse and rabbit IgG (Dianova,Hamburg, Germany). Micrographs were taken with an Olympus AX70microscope equipped with a digital camera (color view 12, soft imagingsystem SiS, Munster, Germany) and analysis software (SiS, Munster,Germany).

Specificity Controls

Method-dependent non-specificities were excluded by running controls asdescribed (Cetin et al., (1994), Proc. Natl. Acad. Sci. USA 91,2935-2939; Cetin et al., (1995) Proc. Natl. Acad. Sci. USA 92,5925-5929). Antibody specificities were tested by preadsorption of theantibodies with homologous and heterologous antigenic peptides (6.25-100mg/ml of the antiserum) (Kulaksiz et al., (2002) Proc. Natl. Acad. Sci.USA 99, 6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664).Preadsorption of the antibodies with homologous antigens atconcentrations as low as 6.25 mg/ml completely blocked immunostaining inthe liver tissues and cells, while preadsorption of the antibodies withheterologous antigens at concentrations up to 100 mg/ml had no effect onimmunostaining.

Hepcidin Elisa Competitive Binding Assay

Determinations were performed in duplicate using 96-well-microtiterplates coated with goat anti-rabbit IgG (DRG Instruments GmbH, Marburg,Germany). Hepcidin antibody EG(2)-IJepN, diluted 1.4000 in Tris bufferedsaline (TBS) containing 40 mM Tris-HCl (pH 7.3), 100 mM NaCl, waspipetted into the microtiter plates. After a 1 hour incubation at roomtemperature (RT), the microtiter plates were washed with TBST (TBS with0.05% Tween 20) and 100 ml standard samples containing various amountsof synthetic peptides or human plasma samples (58 randomized samplesfrom our clinical laboratory) and N-terminally biotinylatedhepcidin-(28-47) (Peptide Specialty Laboratories GmbH, Heidelberg,Germany) (2 ng/well) were added to each well and incubated for 1 hour atRT. The biotinylated antigen-antibody complexes were detected bystreptavidin-peroxidase enzyme (Dako, Hamburg, Germany) with thesubstrate tetramethylbenzidine (DRG); the color reaction was stoppedwith 0.5 N H₂SO₄ and the extinction of the solution was read at 450/630nm wavelength.

Expression of Hepcidin and TfR2 in the Liver and HepG2 Cells

RT-FCR analysis demonstrated that hepcidin is highly expressed in humanliver. Similarly, a 192-bp expected transcript was detected in HepG2cells with an expression level comparable to human liver (FIG. 1). Inaddition, RT-PCR analyses clearly revealed that TfR2 is highly expressedin the human liver and HepG2 cells (FIG. 1). In Western blot analysis,all hepcidin antibodies [EG(1)-HepN, EG(2)-HepN, and EG(1)-HepC]coincidentally identified an immunoreactive band of about 10 kDa inextracts of human and guinea pig liver. This liver peptide comigratedwith an immunoreactive band recognized by a hepcidin antibodies inhomogenates of HepG2 cells (FIG. 1). All antibodies also identified anintensively stained band at −20 kDa in all lanes loaded with human andguinea pig liver extracts or HepG2 cell extracts. Western blot analysisof skeletal muscle extracts (control) showed neither the immunoreactiveband of 10 kDa nor the strong band at 20 kDa (FIG. 1).

Western blot analysis with TfR2 antibody BT-TFR21-S resulted in astaining of an expected (Fleming et al., (2000) Proc. Natl. Acad. Sci.USA 97, 2214-2219) about ˜105 kDa protein in extracts of mouse liver. Inextracts of human liver and HepG 2 cells, a ˜95 kDa immunoreactive TfR2and to lesser extent a ˜105 kda immunoreactive protein was recognized bythe same antibody (FIG. 1). No immunoreactivity was detected in theheart (control tissue).

Cellular and Subcellular Localization of Hepcidin and TfR2

Immunohistochemical studies with various region-specific antibodiesconsistently localized hepcidin to the hepatocytes in human liver (FIG.2). The Kupffer cells, endothelial cells, bile ducts, and the vascularsystem completely lacked hepcidin immunoreactivity. The same antibodiesdetected a strong hepcidin-immunoreactivity also in guinea pig liver(FIG. 2). Interestingly, hepatic lobule, were heterogeneous with respectto a hepcidin immunoreactivity; within a hepatic lobule, a hepcidinimmunoreactive cells were predominantly located in periportal zones, andthe frequency of hepcidin-positive cells continuously decreased from theportal triads toward the central veins (FIG. 3). Notably, distinctintercellular differences exist between a hepcidin positive cells; whilemost hepatocytes were strongly positive for hepcidin, others displayedonly a faint staining or were totally unreactive for hepcidin (FIG. 3).

At the subcellular level, hepcidin immunoreactivity was confined to thebasolateral (=sinusoidal) membrane domain of hepatocytes; noimmunoreactivity was found at the apical membrane domain of therespective cells (FIG. 2). Similarly, immunofluorescence analysisdemonstrated a strong immunoreactivity for hepcidin at the basolateralmembrane domain; immunoreactivity was absent from the apical membranedomain as revealed by double staining with the C219 antibody raisedagainst canalicular P-glycoproteins (Rost et al., (1999) Hepatology 29,814-821).

Corresponding to the localization of hepcidin, protein-specific antibodyBT-TFR21-S detected TfR2 in human and mouse liver. At the cellularlevel, TfR2 was found at the basolateral membrane of hepatocytes, whichrevealed distinct intercellular differences concerning the intensity ofimmunoreactivity (FIG. 4). Heterogeneity was also observed within ahepatic lobule with increasing immunoreactivity from the central veinsto the portal triads.

Immunofluorescence 1N HepG2 Cells

The existence of hepcidin peptide in HepG2 cells was verified byimmunocytochemistry using the corresponding peptide-specific antibodies.All antibodies identified hepcidin by the immunof luorescence techniquein HepG2 cells resulting in a granular immunoreactivity pattern (FIG.5). Coincident with the cellular localization of hepcidin, the TfR2antibody detected TfR2 in the same cells (FIG. 5).

Detection of Hepcidin Propeptide in Human Plasma

Although the C-terminal antibody EG(1)-HepC revealed specific results indot blot, Western blot, immunohistochemistry and immunofluorescenceexperiments (FIGS. 1-5), it did not work in ELISA. The compact foldingpattern of hepcidin and its tertiary structure in the blood may accountfor the inability of the EG(1)-HepC antibody to identify circulatinghepcidin. A sensitive hepcidin ELISA assay with a detection limit of 0.1ng/well of the synthetic peptide was developed with the specificN-terminal hepcidin antibody EG(2)-HepN (FIG. 6). ELISA analyses withthis antibody revealed a high concentration of hepcidin in the rangefrom 5.0 to 308.3 ng per ml human plasma (n=58) (mean.+−.SE; 121.2±73.4.ng/ml). No cross-reactivity was observed when heterologous peptides wereused. As seen in FIG. 6, the ELISA revealed the highest resolving powerbetween 1 and 400 ng/ml, a range, where hepcidin concentrations in humanplasma were determined.

In the present invention, RT-PCR analyses with specific primersconfirmed that hepcidin is highly expressed in the human liver. Threedifferent antibodies recognizing different epitopes in a hepcidinprecursor molecule concurrently identified an immunoreactive peptide ofabout 0.10 kDa by Western blot analysis in liver extracts of twospecies, man and guinea pig. The apparent molecular mass of thisimmunoreactive peptide is in accordance with the molecular mass deducedfor a hepcidin preprohormone from the cDNA sequence (Pigeon et al.,(2001) J. Biol. Chem. 276, 7811-7819). Interestingly, a secondimmunoreactive band of approximately 20 kDa was detected by all hepcidinantibodies in extracts of the human and guinea pig liver but was lackingin the control tissue. This immunoreactive protein may represent ahepcidin-related peptide of higher molecular mass or, because of thetwofold higher molecular mass of the second peptide, it may reflect adimeric type of hepcidin. In fact, in a previous study an aggregationproperty and a possible formation of multimers was described forhepcidin-25 but not for hepcidin-20 (Hunter et al., (2002) J. Biol.Chem., M205305200).

Immunohistochemical and immunofluorescence investigations with threedifferent hepcidin antibodies revealed that, in human and guinea pigliver, hepcidin is specifically localized in hepatocytes mainly locatedaround the portal triads; the coincident staining by differentregion-specific antibodies not only in the human and guinea pig liver,but also in the HepG2 cells (see below) points to hepatocytes being thesource of hepcidin. Hepcidin immunoreactivity decreased from theperiportal zones towards the central veins. This zonation within theportal lobules may have a functional significance, since the periportalhepatocytes have first-pass access to portal veins bringing iron-richblood from the gut. Notably, distinct intercellular differences existbetween hepcidin-positive cells even of the same liver acinus withrespect to the density of hepcidin immunoreactivity that may reflectintercellular differences in expression or secretion of hepcidin.

At the subcellular level, hepcidin was concentrated at the basolateralpole of hepatocytes. No immunoreactivity was found at the apicalmembrane domain. The discrete distribution pattern of hepcidin at thesubcellular level may infer a basolaterally directed release of hepcidininto the liver sinusoids. This directional secretion route isadditionally substantiated by the detection of hepcidin prohormone inhuman plasma (see below); consequently, these findings provide furtherevidence that hepatocytes may regulate iron metabolism in an endocrinefashion via the secretion of the peptide hormone hepcidin.

To analyze the expression and cellular distribution of TfR2 as well asthe respective target membrane domains, RT-PCR, Western blot andimmunohistochemical studies at the cellular level were performed. Asshown in previous studies RT-PCR analyses revealed that TfR2 is highlyexpressed in human liver. (Fleming et al., (2000) Proc. Natl. Acadi.Sci. USA 97, 2214-2219). The presence of this protein was confirmed byWestern blot studies using BT-TFR21-S antibody specific to human andmouse TfR2. A ˜105 kDa immunoreactive protein was detected in mouseliver extracts; this molecular mass of immunoreactive TfR2 is slightlylarger than the expected 95 kDa (Fleming et al., (2000) Proc. Natl.Acadi. Sci. USA 97, 2214-2219) and may represent some posttranslationalmodifications as described previously (Kawabata et al., (2000) J. Biol.Chem. 275, 16618-16625). Under identical conditions, however, theTfR2-antibody identified the protein at the expected 95 kDa molecularmass and with a lower affinity the 105 kDa protein in human liverextracts. The discrepancy between the immunoblots of human and mouseliver may be due to interspecies differences.

Immunohistochemical investigations revealed that TfR2 is localized tohepatocytes of human and mouse liver; coincident with the cellulardistribution of hepcidin, the protein-specific antibody localized TfR2exclusively at the basolateral membrane. This type of membrane-specificassociation of TfR2 argues particularly for a basolateral activation ofTfR2, which is involved in iron metabolism by binding diferrictransferrin and mediating uptake of transferrin-bound iron from theblood into hepatocytes (Philpott, C. C. (2002) Hepatology 35, 993-1001;Subramaniam et al., (2002) Cell Biochem. Biophys. 36, 235-239). Notably,a similar lobular zonation as described for hepcidin was observed forTfR2 with decreasing immunoreactivity from the periportal zones towardthe central veins.

Since an interaction between hepcidin and TfR2 at the cellular level hasbeen discussed in previous studies (Nicolas et al., (2001) Proc. Natl.Acad. Sci. USA 98, 8780-8785; Frazer et al., (2002) Gastroenterology123, 835-844), the coexistence of hepcidin and TfR2 in HepG2 cells-awell-differentiated hepatocellular carcinoma cell line (Aden et al.,(1979) Nature 282, 615-616) was analyzed, demonstrating in many aspectsthe physiology of normal hepatocytes. RT-PCR studies using theappropriate primer specifications and combinations successfully employedin the human liver identified expression of hepcidin and TfR2 in HepG2cells. At the translational level, the presence of hepcidin and TfR2 inHepG2 cells was confirmed by Western blot studies that yieldedimmunoreactive protein bands of correct molecular weights, comigratingwith the corresponding immunoreactive bands from the liver tissues. Theco-localization of the respective proteins in HepG2 cells wasparticularly substantiated by immunocytochemistry using thecorresponding region and molecular domain-specific antibodies. Allantibodies demonstrated hepcidin-labeling in HepG2 cells, revealing agranular immunoreactivity pattern in these cells that inferslocalization of the peptide to small secretory vesicles, alreadydemonstrated in hepatocytes by electron microscopy (Schwartz et al.,(1985) EMBO J. 4, 899-904). TfR2 was immunocytochemically localized,with a peculiar distribution pattern, to HepG2 cells.

On the basis of present data at the transcriptional and translationallevel, hepcidin and TfR2 are coexpressed in the liver and colocalized atthe basolateral membrane domain of hepatocytes. In addition to acoincident localization of TfR2 and hepcidin at the cellular level, asimilar distribution of these molecules within the hepatic lobules witha concentrated immunoreactivity in periportal zones and a decreasingstraining toward the central veins was also detected. The coordinateexpression of these proteins in a common (basolateral) membrane domainand their similar lobular zonation argue for a morphofunctional couplingof the regulating peptide hormonohepcidin and the transferrin-bound ironuptake via TfR2. Indeed, different data substantiate the interactionbetween hepcidin and TfK2. First, alterations in transferrin saturation,probably sensed by TfR2, modulate the expression of hepatic hepcidin(Philpott, C. C. (2002) Hepatology 35, 993-1001). Second, as revealedfrom quantitative RT-PCR analyses on human liver, hepatic expression ofTfR2 correlates significantly with hepcidin expression regulated by thetransferrin saturation (S. G. Gehrke, H. Kulaksiz et al. unpublisheddata). Third, hepcidin and TfR2 are colocalized at a common cellmembrane domain and reveal the same lobular distribution with a strongimmunoreactivity in periportal zones, the site, where in case ofmutations that abrogate expression of TfR2 (Fleming et al., (2002) Proc.Natl. Acad. Sci. USA 99, 10653-10658) and hepcidin (Nicolas et al.,(2001) Proc. Natl. Acad. Sci. USA 98, 8780-8785) but also hepcidin (Zhouet al., (1998) Proc. Natl. Acad. Sci. USA 95, 2492-2497; Levy et al.,(1999) Blood 94, 9-11) and B2m (Santos et al., (1996) J. Exp. Med. 184,1975-1985) hepatic iron overloading occurs. The clinical consequences ofiron overload include cirrhosis of the liver and hepatocellular cancer,diabetes, heart failure, arthritis, and hypogonadism. Zhou et al., Proc.Natl. Acad. Sci., 95, 2492-2497 (1998). Fourth, mutations in the TfR2gene were reported to lead to hemoechromatosis (Camasehella et al.,(2000) Nat. Genet. 25, 14-15); this may result from decreased hepcidinexpression, which, in turn, results in increased iron absorption(Nicolas et al., (2001) Proc. Natl. Acad. Sci. USA 98, 8780-8785).Increased iron absorption in patients suffering from hereditaryhemochromatosis leads to accumulation of iron, with eventual tissuedamage and organ disfunction. When the disorder remains untreated,premature mortality resulting from hepatocellular carcinoma, cirrhosis,cardiomyopathy, or diabetes mellitus is common. Santos et al., (1996) J.Exp. Med. 184, 1975-1985.

Since blood-forming tissues and sites of iron storage, such as theliver, are thought to transmit signals to the intestinal cells thatindicate the body's requirements for dietary iron (Philpott, C. C.(2002) Hepatology 35, 993-1001), hepcidin is a candidate signalingfactor secreted from the liver and regulating the intestinal ironabsorption. However, there is still controversy about the existence ofcertain molecular forms of hepcidin in the blood (Krause et al., (2000)FEBS Lett. 480, 147-150; Park et al., (2001) J. Biol. Chem. 276,7806-7810; Hunter et al., (2002) J. Biol. Chem., M205305200). To analyzewhether the prohormone of hepcidin is secreted into the blood, and toassess the range of hepcidin concentration in human plasma, an ELISA wasdeveloped by applying the same N-terminal antibody against hepcidinprohormone used successfully in Western blot, immunocytochemical andimmunofluorescence experiments. The ELISA was characterized by a highsensitivity with a detection limit of 0.1 ng/well and a powerfulresolution in the range of 1 to 400 ng/ml; the range, where hepcidinconcentrations were determined. In the human plasma samples tested(n=58), a high concentration of pro-hepcidin (mean+SE 121.2.+−.73.4ng/ml) was measured, ranging from 5.0 to 308.3 ng/ml, which iscomparable with the concentration of known regulating peptide hormonesand approximately 1.2-fold higher than the concentration of hepcidin inhuman urine (Park, C. H., Valore, E. V., Waring, A. J. & Ganz, T. (2001)J. Biol. Chem. 276, 7806-7810). Interestingly, the measuredconcentrations exhibit a wide range of pro-hepcidin indicating that thepeptide may be subject to strong regulation. Future experiments areintended to determine hepcidin concentrations in plasma of varioussubjects with disturbances of iron metabolism and to analyze themolecular mechanism of hepcidin regulation using the established ELISA.

The cDNA structure suggests that hepcidin is translated as an 84 aminoacid prepropeptide that is N-terminally processed to a 20-25 amino acidpeptide (id.). Although a strong consensus sequence for a signalsequence cleavage site is located between Gly24 and Ser.sup.25 thatwould result in a 60 residue propeptide, previous studies failed toisolate the larger propeptide from native sources like liver tissue andblood (Id.). In addition to technical difficulties, the abundance ofpropeptide convertases in the liver may inhibit the isolation of certainpropeptides. In this context, recent studies have shown that the humancirculating form of hepcidin described by two research groups in blood(Krause et al., (2000) FEBS Lett. 480, 147-150) and in urine (Park etal., (2001) J. Biol. Chem. 276, 7806-7810), consists of the C-terminal20-25 amino acids of the protein. However, the ELISA measurements of thepresent invention were performed with the specific-antibody raisedagainst the N-terminus of hepcidin precursor, implying that besides the20-25 amino acid processed forms, a hepcidin prohormone is secreted andcirculates in human blood.

To understand the role of hepcidin, the knowledge about the cellularorigin and the signaling pathway of the peptide is necessary. In thisrespect, the present invention describes hepcidin immunoreactivity inhuman and guinea pig liver, where it is localized to the basolateralmembrane domain of hepatocytes. Previous studies have speculated on apossible connection between these cells and the absorptive enterocytes(Hunter et al., (2002) J. Biol. Chem., M205305200; Anderson et al.,(2002) Biochem. Soc. Trans. 30, 724-726). The present inventiondescribes the detection of pro-hepcidin in the human plasma therebyindicating that hepatocytes secrete the prohormone of hepcidin that maydecrease dietary iron absorption via an endocrine pathway. Moreover,hepcidin was detected in HepG2 cells, where the newly discoveredtransferrin receptor type 2 was also found. The simultaneous existenceof hepcidin and TfR2 in HepG2 cells and their common polarizedlocalization and lobular distribution in the liver may indicate thathepcidin is an intrinsic hepatic peptide morphofunctionally coupled toTfR2, which is regulated by transferrin saturation and, in turn,modulates expression of hepcidin. Hence, pertinent findings are expectedfrom studies on the signaling pathway of hepcidin.

Enzyme Immunoassay for the Quantitative Measurement of Hepcidin in Humanor Animal Serum and Other Body Fluids.

In one embodiment of the invention a Hepcidin enzyme immunoassay (“EIA”)is used. An EIA is a solid phase enzyme-linked immunosorbent assay(ELISA) based on the competitive principle. Microtiter wells of a 96well microtiter plate are coated with a polyclonal rabbit anti-hepcidinantibody. An unknown amount of Hepcidin present in the sample and afixed amount of Hepcidin conjugated with a biotin molecule compete forthe binding sites of the Hepcidin antibodies immobilized on the wells.After one hour incubation the microtiter plate is washed to stop thecompetition reaction. In the following incubation the bound biotinmolecules are detected with streptavidin horseradish peroxidase. Afterone half hour of incubation the plate is washed a second time. Havingadded the substrate solution the concentration of Hepcidin is inverselyproportional to the optical density measured.

Materials

-   -   1. Microtiter wells.    -   wells coated with Anti-Hepcidin antibody (96 wells).    -   2. Reagent: Biotin Conjugate (Hepcidin conjugated to biotin) 7        ml.    -   3. Reference Standard Set, 1.0 ml each    -   0, 20, 100, 500, 1000 ng/ml.    -   4. Reagent: Enzyme Complex (Streptavidin conjugated to        horseradish peroxidase (“HRP”)) 14 ml.    -   5. Reagent: Substrate Solution-HS-TMB, 14 ml.    -   6. Stop Solution, 0.5M H₂SO₄, 14 ml.    -   7. Wash Solution, 40×,30 ml.    -   8. A microtiterplate reader (450±10 nm) (e.g., the DRG        Instruments Microtiterplate Reader).    -   9. Precision micropipettes with disposable tips for 50 and 100        μl.    -   10. Standard refrigerator.    -   11. Absorbent paper.    -   12. Deionized water.

While this embodiment has been described in terms of preferredmaterials, a person skilled in the art of the invention will appreciatethat other materials can be used in the invention. For example, one ofskill in the art will appreciate that complementary binding moietiesother than biotin/streptavidin, as well as enzyme/substrate combinationsother than horse radish peroxidase/peroxide, may be used in theinvention.

Storage Conditions

When stored at 2° to 8° C. unbroken reagents will retain reactivityuntil expiration date. Do not use reagents beyond this date. Microtiterwells must be stored at 2° to 8° C. Once the foilbag has been brokencare should be taken to close it tightly again. The immuno-reactivity ofthe coated microtiter wells is stable for approximately 6 weeks in thebroken, but tightly closed plastic zip pouch containing the desiccant.

Specimen Collection and Preparation

Human or animal serum or EDTA plasma should be used in the assay. Nospecial pretreatment of the biological sample is necessary. Thebiological sample may be stored at 2-8° C. for up to 24 hours, andshould be frozen at −20° C. or lower for longer periods. Do not usegrossly hemolyzed or grossly lipemic specimens. For other samplematerial a special extraction protocol may be necessary.

Performance of the Assay

General Remarks:

1. All reagents and specimens must be allowed to come to roomtemperature before use. All reagents must be mixed without foaming.

2. Once the test has been started, all steps should be completed withoutinterruption.

3. Use new disposable plastic pipette tips for each reagent, standard orspecimen in order to avoid cross contamination. For the dispensing ofthe Substrate Solution and the Stop Solution avoid pipettes with metalparts.

4. Pipette standards and samples onto the bottom of the well. Forpipetting of Enzyme Conjugate and Stop Solution it is recommended tohold the pipette in a vertical position above the well and dispense thecorrespondent solution into the center of the well so that a completemixing of Enzyme Conjugate with sample or standard and of the StopSolution with the Substrate Solution is achieved.

5. Before starting the assay, it is recommended that all reagents beready, caps removed, all needed wells secured in holder, etc. This willensure equal elapsed time for each pipetting step without interruption.

6. As a general rule the enzymatic reaction is linearly proportional totime and temperature. This makes interpolation possible for fixedphysico-chemical conditions. If in a test run the absorbance of ZeroStandard is lower than 1,0 or above the upper performance limit of yourmicrotiterplate spectrophotometer you can extend or reduce theincubation time of the final enzymatic formation of color to 30 or 10minutes accordingly. Since calibrators are assayed in each run,absorbance fluctuations do not affect the result.

7. The Substrate Solution should be colorless or slightly blue or green.If the solution is dark blue the reagent is unusable and must bediscarded.

8. During incubation with Substrate Solution avoid direct sunlight onthe microtiter plate.

Reagent Preparation

Wash Solution: Add deionized water to the 40× concentrated Wash Solution(contents: 30 ml) to a final volume of 1200 ml. The diluted WashSolution is stable for 2 weeks at room temperature.

Assay Procedure

-   -   1. Secure the desired number of coated strips in the holder.    -   2. Dispense 50 μl of Hepcidin Standards into appropriate wells.    -   3. Dispense 50 μl of sample into selected wells.    -   4. Dispense 50 μl of Biotin Conjugate into each well.    -   5. Thoroughly mix the plate for 10 seconds. It is important to        have complete mixing in this step.    -   6. Incubate for 60 minutes at room temperature.    -   7. Briskly shake out the contents of the wells.    -   8. Rinse the wells 3 times with diluted Wash Solution (400 μl        per well). Strike the wells sharply on absorbent paper to remove        residual droplets.    -   9. Add 100 μl Streptavidin HRP Complex to all wells.    -   10. Incubate for 30 minutes at room temperature.    -   11. Briskly shake out the contents of the wells.    -   12. Rinse the wells 3 times with diluted Wash Solution (400 μl        per well). Strike the wells sharply on absorbent paper to remove        residual droplets.    -   13. Add 100 μl of Substrate Solution to each well, at timed        intervals.    -   14. Incubate for 15 minutes at room temperature.    -   15. Stop the enzymatic reaction by adding 100 μl of Stop        Solution to each well at the same timed intervals as in step 10        and determine the absorbance of each well at 450±10 nm.        Final Reaction Stability

It is recommended that the wells be read within 30 minutes followingstep 15.

Calculation of Results

Any microwell reader capable of determining the absorbance at 450±10 nmmay be used. The Testosterone value of each sample is obtained asfollows:

-   -   1. Using linear-linear or semi log graph paper, construct an        standard curve by plotting the average absorbance (Y) of each        Reference Standard against its corresponding concentration (X)        in ng/ml. For construction of the standard curve we recommend a        four parameter logistic function.    -   2. Use the average absorbance of each sample to determine the        corresponding Testosterone value by simple interpolation from        this standard curve, multiplying by the initial sample dilution,        if necessary.

A DRG ELIZA MAT 3000 and the DRG Regression Program allow the readingand computer assisted interpretation using a four parameter logisticfunction.

INDUSTRIAL APPLICABILITY

The invention has applications in connection with diagnosing a diseasecondition characterized by non-physiological levels of hepcidin protein,including prohepcidin and fragments thereof.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

All patent and non-patent publications cited in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. All these publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated herein by reference.

1. A kit for detecting a level of a hepcidin precursor, the kit comprising: an anti-hepcidin precursor antibody or fragment thereof that specifically binds to one or more epitopes of the hepcidin precursor amino acid sequence located within amino acids 25-49 of SEQ ID NO: 2, and a reagent that binds directly or indirectly to the antibody or fragment thereof, and wherein the antibody or fragment thereof detects the hepcidin precursor by Western blot analysis in a tissue or a liquid sample obtained from a subject.
 2. The kit of claim 1 wherein the anti-hepcidin precursor antibody or fragment thereof is immobilized on a support.
 3. The kit of claim 2 wherein the reagent comprises the hepcidin precursor complexed with a first binding molecule.
 4. The kit of claim 3 wherein the first binding molecule is biotin.
 5. The kit of claim 4 wherein the kit further comprises an enzyme complexed with a second binding molecule and a substrate of the enzyme.
 6. The kit of claim 5, wherein the second binding molecule is streptavidin.
 7. The kit of claim 5, wherein the enzyme is horse radish peroxidase, and the substrate comprises peroxide.
 8. An isolated antibody or fragment thereof that specifically binds to one or more epitopes of a hepcidin precursor amino acid sequence located within amino acids 25-49 of SEQ. ID. NO. 2, and wherein the antibody or fragment thereof detects an amino acid sequence consisting of amino acids 25-84 of SEQ ID NO: 2 by Western blot analysis in a tissue or a liquid sample obtained from a patient. 